A 120 nucleotide bacteriophage PHI29 RNA is structural and catalytic constituent of the viral precursor shell (prohead) into which the 18 kilobase pair double-stranded viral DNA is packaged in an efficient, ATP-dependent, in vitro reaction. The RNA is a part of the portal vertex of the prohead (connector), which together with the PHI29 DNA packaging protein gp16 may function as an ATPase needed for translocation of DNA-gp3 (PHI29 DNA has a covalently bound protein gp3 at each 5' terminus). Thus the PHI29 DNA "packaging machine" includes the connector protein (gp10), RNA, gp16, and perhaps the gp3 of DNA-gp3. The role of the RNA in the sequential initiation and translocation events of in vitro DNA-gp3 packaging and its possible function as part of the ATPase will be investigated. Localization of the RNA in the connector will be attempted by processing of electron micrographs and three-dimensional reconstructions. Prohead RNAs of PHI29 relatives and other phages will be isolated and sequenced. The higher-order structure of PHI29 prohead RNA and counterpart RNAs will be studied by the use of comparative sequence analysis, footprinting, and chemical and enzymatic probes.